Abstract
A universal tool in the culture-based study of bacterial viruses (bacteriophages, or phages) is the agar overlay, which is used in the isolation of new viruses, and in their quantification and purification. Here, simple optimizations that increase efficiency and throughput in agar overlay based isolation and cultivation of virus-host systems are presented. The agar overlay is streamlined to minimize steps and materials. Serial purification of viruses from viral colonies (plaques) is optimized to eliminate steps by combining purification by serial re-streaking with the optimized agar overlay approach. Finally, recommendations are made for efficient archival and storage of virus plaques. In sum, this work presents:•Tube-free Agar Overlays: rapid plaque assays with fewer steps and materials•Molten Streaking for Singles: rapid tube-free serial purification of viruses•Archiving Plaques: saving virus purification for later
Highlights
Method ArticleABSTRACTA universal tool in the culture-based study of bacterial viruses (bacteriophages, or phages) is the agar overlay, which is used in the isolation of new viruses, and in their quantification and purification
Background and applicationsThis protocol simplifies current approaches for agar overlay plaque assays by eliminating the use of tubes for premixing of agar, hosts, and viruses, in favor of pipetting each of these directly onto the bottom agar
The development of a method for improving plaque assays by incorporating viruses and hosts into a low density ‘top’ agar layered upon a higher density ‘bottom’ agar layer was described by André Gratia in 1936 [22,23], and by Alfred Hershey et al in 1943 [2]
Summary
ABSTRACTA universal tool in the culture-based study of bacterial viruses (bacteriophages, or phages) is the agar overlay, which is used in the isolation of new viruses, and in their quantification and purification. Simple optimizations that increase efficiency and throughput in agar overlay based isolation and cultivation of virus-host systems are presented. The agar overlay is streamlined to minimize steps and materials. Serial purification of viruses from viral colonies (plaques) is optimized to eliminate steps by combining purification by serial restreaking with the optimized agar overlay approach. Recommendations are made for efficient archival and storage of virus plaques. ARTICLE INFO Method name: Agar overlay Keywords: Agar overlay, Agar layer, Top agar, Bottom agar, Plaque, Virus, Phage, Isolation, Cultivation, Purification Article history: Received 25 October 2017; Accepted 16 January 2018; Available online 31 January 2018
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